Development of multiplex PCR assay for detection of Alternaria brassicae, A. brassicicola and Xanthomonas campestris pv. campestris in crucifers.

Among biotic stresses, Alternaria leaf spots caused by Alternaria brassicae and A. brassicicola and black rot caused by Xanthomonas campestris pv. campestris are major limiting factors in brassica cultivation across the world. Because of seed-borne nature of these pathogens primarily, disease-free conservation as well as exchange of brassica seeds at domestic as well as international level are major challenges. To facilitate disease-free conservation and transboundary movement of brassica germplasm, a highly specific and sensitive method was developed for simultaneous detection of these pathogens. A set of primers namely, AbeABC1F and AbeABC1R based on ABC transporter (Atr1) gene for A. brassicae, Aba28sF and Aba28sR based on SSR marker was developed for A. brassicicola as well as rpf gene-based primers namely, rpfH_F and rpfH_R for X. campestris pv. campestris were used for multiplex PCR. The specific bands of 586, 201 and 304 bp were obtained in multiplex PCR assay for A. brassicae, A. brassicicola and X. campestris pv. campestris, respectively. Therefore, the developed multiplex PCR protocol could be utilized for a reliable diagnosis of these pathogens to facilitate safe conservation, exchange of seeds to the researchers and also by seed certification agencies for ensuring quality seed availability to farmers.